Vibrio cholerae 0139 conjugate vaccines

ABSTRACT

The invention pertains to conjugates of the capsular polysaccharide of  Vibrio cholerae  O139, or a structurally and/or immunologically related oligo- or poly-saccharide, and a carrier. These conjugates are useful as pharmaceutical compositions and/or vaccines to induce serum antibodies which have bactericidal (vibriocidal) activity against  V. cholerae , in particular  V. cholerae  O139, and are useful to prevent, treat and/or reduce the severity of disease caused by  V. cholerae  infection, such as cholera. The present invention also relates to diagnostic tests for  V. cholerae  infection, and/or cholera caused by  V. cholerae  infection, using one or more of the oligo- or poly-saccharide-carrier conjugates or antibodies described above.

PRIORITY CLAIM

This is a § 371 U.S. national stage of PCT/US00/24119, filed Sep. 1,2000, which was published in English under PCT Article 21(2).

FIELD OF THE INVENTION

This invention relates to compositions and methods for eliciting animmunogenic response in mammals, including responses which provideprotection against, or reduce the severity of, bacterial infections.More particularly it relates to conjugates of the capsularpolysaccharide of Vibrio cholerae O139, or a structurally and/orimmunologically related oligo- or poly-saccharide, and a carrier. Theseconjugates are useful as pharmaceutical compositions and/or vaccines toinduce serum antibodies which have bactericidal (vibriocidal) activityagainst V. cholerae, in particular V. cholerae O139, and are useful toprevent, treat and/or reduce the severity of disease caused by V.cholerae infection, such as cholera.

The present invention also relates to diagnostic tests for V. choleraeinfection, and/or cholera caused by V. cholerae infection, using one ormore of the oligo- or poly-saccharide-carrier conjugates, or antibodiesdescribed above.

The present invention also relates to methods of making oligo- orpoly-saccharide carrier conjugates using CDAP to activate carboxylicacids on the carbohydrate. The present invention also relates to methodsof separating V. cholerae CPS from contaminating smaller molecules bymeans of diafiltration.

Abbreviations used: LPS: lipopolysaccharide; CPS: capsularpolysaccharide; O-SP: O-specific polysaccharide; DT: diphtheria toxin;HSA: human serum albumin; DCC: dicyclohexyl carbodiimide; EDC:1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; CDAP:1-cyano-4-dimethylaminopyridinium tetrafluoroborate; ADH: adipic aciddihydrazide; rDT: recombinant diphtheria toxin mutant CRMH21G.

BACKGROUND OF THE INVENTION

The most successful of all carbohydrate pharmaceuticals so far have beenthe carbohydrate-based antibacterial vaccines [48]. The basis of usingcarbohydrates as vaccine components is that the capsular polysaccharidesand the O-specific polysaccharides on the surface of pathogenic bacteriaare both protective antigens and essential virulence factors. The firstsaccharide-based vaccines contained capsular polysaccharides ofPneumococci: in the United States a 14-valent vaccine was licensed in1978 followed by a 23-valent vaccine in 1983. Other capsularpolysaccharides licensed for human use include a tetravalentmeningococcal vaccine and the Vi polysaccharide of Salmonella typhi fortyphoid fever. The inability of most polysaccharides to elicitprotective levels of anti-carbohydrate antibodies in infants and adultswith weakened immune systems can be overcome by their covalentattachment to proteins that confer T-cell dependent properties [49].This principles has led to the construction of vaccines againstHaemophilus influenzae b (Hib) [37] and in countries where thesevaccines are routinely used, meningitis and other diseases caused by Hibhave been virtually eliminated [50]. Extension of the conjugatetechnology to the O-specific polysaccharides of Gram-negative bacteriahas provided a new generation of glycoconjugate vaccines that areundergoing various phases of clinical trials [51].

Cholera remains an important public health problem. The long-termcontrol of cholera depends on good personal hygiene, uncontaminatedwater supply and appropriate sewage disposal. However, the improvementof hygiene is a distant goal for many countries. Thus the availabilityof an effective cholera vaccine is important for the prevention ofcholera in these countries. Research on new cholera vaccines has mainlyfocused on oral formulations that stimulate the mucosal secretory immunesystem. Two oral cholera vaccines have been experimented with on largescale in humans.

The first vaccine, containing inactivated bacterial cells and theB-subunit of cholera toxin, was tested in Bangladesh from 1985 to 1989.This vaccine, according to the WHO, may prove useful in the stable phaseof refugee/displaced person crises, especially when given preventively.The second vaccine is a live attenuated vaccine containing thegenetically manipulated V. cholerae O1 strain CVD 103-HgR. Despite itsefficacy in adult volunteers, results of a large-scale field trialcarried-out in Indonesia for 4 years have shown a surprisingly lowprotection. Moreover, one of the safety concerns associated with livecholera vaccine is a possible horizontal gene transfer and recombinationevent leading to reversion to virulence. [52]

More recently, conjugates of V. cholerae O1 lipopolysaccharide withcholera toxin variants were prepared with an adipic acid dihydrazidelinker. In Phase I studies, these conjugates elicited vibriocidalantibodies in human volunteers, with IgM levels comparable to, and IgGlevels superior to, the Ig levels elicited by a cellular vaccine [10,36, 38, 39]. Conjugation of the V. cholerae O139 CPS to tetanus toxoid,and inoculation of mice with the conjugate, has also been described byMorris et al. in U.S. Pat. No. 5,653,986.

Until 1992, V. cholerae serogroup O1 was recognized as the sole cause ofcholera epidemics, whereas the non-O1 serogroups were associated withsporadic cases of gastroenteritis and extra-intestinal infections. Inlate 1992, the etiological agent of a massive cholera epidemic wasidentified as non-O1 V. cholerae serogroup O139. [53] This was the firstreported instance of an encapsulated strain that caused epidemiccholera. [11]

The surface polysaccharide of V. cholerae O1 is a lipopolysaccharide(LPS), whereas V. cholerae O139, in contrast, has a capsularpolysaccharide (CPS) composed of a hexasaccharide repeating unit,containing a trisaccharide backbone and two branches [3, 4, 8, 11, 16,17, 19, 26, 28, 30, 31, 35, 38, 42, 43, 45]. The repeating unit containstwo negatively charged groups, a galacturonic acid carboxyl group and acyclic phosphate diester. This repeating unit is incorporated into theV. Cholerae O139 lipopolysaccharide as well as the capsularpolysaccharide, and it is possible that the V. cholerae O139 CPS is infact a very high molecular weight LPS.

Passive immunization of mice with antiserum to the V. cholerae O139capsular polysaccharide has been shown to protect against variants of V.cholerae O139, and it has been proposed that conjugates of the V.cholerae O139 capsular polysaccharide with cholera toxin or toxoid mightbe “worthy of further study” [38]. A potential live oral vaccine,comprising a non-pathogenic deletion mutant of V. cholerae engineered toexpress the V. cholerae O139 capsular polysaccharide and core-linkedO-polysaccharide, has been described [62]. The vaccine elicited anti-CPSantibodies in rabbits, but neither animal protection studies norclinical results have been reported to date.

It has been proposed that a critical level of serum IgG to the surfacepolysaccharides of V. cholerae O1 and V. cholerae O139 confersserotype-specific immunity to cholera [3, 7, 17, 24, 25, 28, 29-32, 38,39, 43, 44]. It has also been proposed that the level of IgG, ratherthan the total level of vibriocidal antibodies may correlate moreaccurately with protection against cholera, because (1) synthesis of IgGis predictive of long-lived immunity, probably reflecting induction ofT-helper cells to the antigen-specific B-cells, and (2) IgG antibodiespenetrate into the extracellular spaces and interior of the smallintestine more effectively than IgM. IgG directed to the O-specificpolysaccharide of V. cholerae O1 or V. cholerae O139 could conferprotective immunity to cholera by inactivating the inoculum on theintestinal mucosal surface.

Currently, vibriocidal antibody titers induced by vaccines are regardedas being predictive of therapeutic utility, at least for vaccines thathave passed regulatory review: vibriocidal titer is the only serologicassay required by the U.S. Food and Drug Administration for licensure ofnew cholera vaccine lots. [61].

Previously described conjugates of the V. cholerae O139 CPS have notdemonstrated the induction of adequate levels of IgG antibodies toprovide reliable vaccines; accordingly there still remains a need forimproved conjugates.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide conjugatescomprising the capsular polysaccharide of V. cholerae O139 and acarrier. It is also an object of the invention to provide conjugatescomprising oligo- or poly-saccharides which are structurally relatedand/or antigenically similar to the capsular polysaccharide of V.cholerae O139. Preferably, these oligo- or poly-saccharides of theinvention are antigenically similar to the capsular polysaccharide of V.cholerae O139. These oligo- or poly-saccharide conjugates areimmunogenic and elicit serum antibodies that are bactericidal against V.cholerae, in particular V. cholerae O139, and are useful in theprevention, treatment, and reduction in severity of disease caused by V.cholerae. These oligo- or poly-saccharide conjugates, and the antibodieswhich they elicit, are also useful for studying V. cholerae, inparticular V. cholerae O139, in vitro, and for studying its products inpatients.

It is yet another object of the present invention to provide antibodieswhich have vibriocidal activity against V. cholerae, in particular V.cholerae O139, and which react with, or bind to, the capsularpolysaccharide of V. cholerae O139, wherein the antibodies are elicitedby immunization with a carrier-conjugate comprising the natural V.cholerae capsular polysaccharide, or a structurally and/orimmunologically related natural, synthetic or semi-synthetic oligo- orpoly-saccharide, preferably a semi-synthetic or synthetic oligo- orpoly-saccharide comprising one or more, preferably four or more,repeating hexasaccharide units of V. cholerae O139 capsularpolysaccharide.

It is yet another object of the present invention to providecarrier-conjugates which are useful as pharmacuetical compositionsand/or vaccines to prevent, treat and/or ameliorate diseases, such ascholera, caused by V. cholerae, in particular V. cholerae O139.

It is yet another object of the present invention to prepare antibodiesfor the prevention, treatment or amelioration of cholera. Antibodieselicited by the carrier conjugates of the invention are useful inproviding passive protection to an individual exposed to V. cholerae, inparticular V. cholerae O139, to prevent, treat, or ameliorate infectionand disease caused by the microorganism.

It is yet another object of the present invention to provide diagnostictests and/or kits for disease caused by V. cholerae, in particular V.cholerae O139, using one or more of the carrier-conjugates, and/orantibodies, of the present invention.

It is yet another object of the present invention to provide a methodfor synthesizing a conjugate vaccine comprising V. cholerae O139capsular polysaccharide covalently linked to a polypeptide, such asdiphtheria toxin (DT) derivative which has a lower toxicity than DT andis suitable for clinical use.

Methods are also provided to conjugate the natural, semi-synthetic, orsynthetic oligo- or poly-saccharides of the invention with a carrier.

Methods are also provided for separating CPS from contaminating smallermolecules by diafiltration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SEPHAROSE™ (cross-linked agarose) CL-4B gel filtration profileof the unfractionated V. cholerae O139 capsular polysaccharide (CPS).Refractive index (RI) response (broken line); Colitose-containingfractions (solid line); the arrow indicates the point of separationbetween the retentate and filtrate by diafiltration (Amicon YM100) ofthe unfractionated CPS; Distribution coefficients (Kd) are depictedabove each peak.

FIG. 2. ¹³C NMR spectrum of V. cholerae O139 capsular polysaccharide.Legend: ¹³C NMR spectrum of the CPS (50 mg/ml D₂O) was measured usingVarian XL3000 spectrometer by averaging 50,000 scans with a 10-s decaybetween acquisition and 10 μs 90° pulse. Prior to Fouriertransformation, a 5-Hz line broadening was applied and zero-filled to32,000 datum points.

FIG. 3. SEPHAROSE™ (cross-linked agarose) CL-4B gel filtration profilesof V. cholerae O139 CPS conjugates with rDT. Representativechromatograph of the CPS_(AH)-rDT conjugates (A) prepared byEDC-mediated synthesis or of CPS-rDT_(AH) conjugates (B) prepared byCDAP-mediated synthesis. Legend: Polysaccharide (▪) and protein (∘).

FIG. 4. Double immunodiffusion of V. cholerae O139 conjugates withmurine hyperimmune cholera O139 and equine diphtheria toxin antisera:(A) representative pattern for CPS_(AH)-rDT conjugates and (B)representative pattern for CPS-rDT_(AH) conjugates. Legend: 1 murinehyperimmune cholera O139 antiserum, 3 μl; 2 equine diphtheria toxinantiserum, 5 μl, 3 conjugate (PS: 1-4 μg; PR: 1-8 μg).

FIG. 5. Structure of the repeating hexasaccharide unit of the V.cholerae O139 capsular polysaccharide.

DETAILED DESCRIPTION OF THE INVENTION

In preliminary studies the present inventors found that V. cholerae CPSdoes not elicit serum antibodies after three injections in mice. Toimprove its immunogenicity, CPS was covalently bound by a variety ofdifferent synthetic methods to chicken serum albumin, a model protein.The resultant conjugates induced serum anti-CPS IgG in mice withvibriocidal activity. CPS was then covalently bound by several methodsto the diphtheria toxin mutant CRMH21G.

The recombinant diphtheria toxin mutant CRMH21G was prepared byreplacing histidine 21 with glycine in the A-chain of diphtheria toxin[15]. This mutant protein has a 1×10⁻⁴ lower toxicity than diphtheriatoxin (DT) and is suitable for clinical use. The two synthetic schemesfound most successful with the chicken albumin, involving1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activatingagents, were adapted to prepare 4 conjugates of V. cholerae O139 CPSwith the recombinant diphtheria toxin mutant, CRMH21G. Adipic aciddihydrazide was used as a linker.

When injected subcutaneously into young outbred mice in a clinicallyrelevant dose and schedule, these conjugates elicited very high levelsof serum CPS antibodies of IgG and IgM classes, with vibriocidalactivity to strains of capsulated V. cholerae O139. Treatment of thesesera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, theirvibriocidal activity. These results indicate that the conjugateselicited IgG with vibriocidal activity. The conjugates also elicitedhigh levels of serum diphtheria toxin IgG.

Convalescent sera from 20 cholera patients infected with V. choleraeO139 had vibriocidal titers ranging from 100 to 3200. Absorption withthe CPS reduced vibriocidal titer of all sera to ≦50. Treatment with2-ME reduced the titers of 17 of the 20 to ≦50. These data show that,similar to infection with V. cholerae O1, infection with V. choleraeO139 induces vibriocidal antibodies specific to the surfacepolysaccharide of this bacterium (CPS) that are mostly of IgM class.

These results clearly indicate that the conjugates of the invention arecapable of inducing anti-V. cholerae CPS antibodies having desirableproperties. Based on these data, clinical trials of the V. cholerae O139CPS-rDT conjugates of this invention are planned.

Accordingly, one object of the invention is a vaccine that will induceantibodies with vibriocidal activity against V. cholerae, in particularV. cholerae O139. These antibodies may be obtained by parenteraladministration of a vaccine containing natural V. cholerae CPS, or astructurally and/or immunologically related natural, synthetic orsemi-synthetic oligo- or poly-saccharide, conjugated to a carrier. Theoligo- or poly-saccharide, as a natural, synthetic, or semi-syntheticproduct, may be bound to both a carrier saccharide and a non-toxicnon-host protein carrier or directly to a non-toxic non-host proteincarrier to form a conjugate. The present invention also encompassesmixtures of the oligo- or poly-saccharides and conjugates thereof.

The vaccine compositions of the invention will preferably induceprotective levels of anti-V. cholerae O139 antibodies, so as to renderthe recipient immune to infection by V. cholerae O139, or resistant tocholera caused by V. cholerae O139, after one or more doses of vaccine.The levels of antibodies induced by the vaccine will preferably resultin vibriocidal titers of greater than 800, more preferably greater than1600, and most preferably greater than 3200, when measured against V.cholerae O139 SPH1168.

The saccharide-based vaccine is intended for active immunization forprevention of cholera, but may also be used for preparation of immuneantibodies as a therapy. This CPS-based vaccine is designed to conferspecific preventative immunity to infection with V. cholerae, inparticular V. cholerae O139, and to induce antibodies specific to V.cholerae O139 CPS for prevention and/or treatment of cholera.

The conjugates of the invention, as well as the antibodies thereto, willbe useful in increasing resistance to, preventing, ameliorating, and/ortreating disease, such as cholera, caused by V. cholerae, in particularV. cholerae O139, in humans.

Specifically, it is expected that conjugates of V. cholerae O139 CPSwill elicit serum antibodies specific to V. cholerae O139 CPS, whichshould induce complement-dependent killing of V. cholerae O139. It isalso expected that these serum antibodies specific to V. cholerae O139CPS will protect against V. cholerae O139, infection in mammals,including humans.

A number of primary uses for the compounds of this invention areenvisioned. The invention is intended to be included in the routineimmunization schedule of infants and children living in areas wherecholera is endemic, and in individuals at risk for cholera, such astravelers to areas where cholera is endemic. It is also intended to beused for intervention in epidemics caused by V. cholerae O139.Additionally, it is planned to be used for a multivalent vaccine for V.cholerae and other enteric pathogens for routine immunization ininfants.

The invention may also be used to prepare antibodies with vibriocidalactivity against V. cholerae, in particular V. cholerae O139, fortherapy of cholera. The invention may also be used to provide adiagnostic test for cholera caused by V. cholerae, in particular V.cholerae O139.

The conjugates of the invention are also expected to be capable ofinducing anti-DT antibodies which may prevent, lessen or attenuate theseverity, extent or duration of an infection by Corynebacteriumdiptheriae.

Definitions:

“Oligosaccharide” as defined herein is a carbohydrate containing up totwelve monosaccharide units linked together. A “polysaccharide” asdefined herein is a carbohydrate containing more than twelvemonosaccharide subunits linked together.

As used herein, “natural” refers to a native or naturally occurringoligo- or poly-saccharide which has been isolated from an organism,e.g., V. cholerae O139, and “semi-synthetic” refers to a native ornaturally occurring polysaccharide that has been structurally altered.Such structural alterations are any alterations that render the modifiedpolysaccharide antigenically similar to the capsular polysaccharide ofV. cholerae, in particular V. cholerae O139. Preferably, the structuralalterations substantially approximate the structure of an antigenicdeterminant of the capsular polysaccharide of V. cholerae O139.

In other words, a modified oligo- or poly-saccharide of this inventionis characterized by its ability to immunologically mimic the capsularpoly-saccharide of V. cholerae O139, in particular V. cholerae O139.Such a modified oligo- or poly-saccharide is useful herein as acomponent in an inoculum for producing antibodies that preferablyimmunoreact with, or bind to, the capsular polysaccharide of V. choleraeO139.

As used herein, the term “immunoreact” means specific binding between anantigenic determinant-containing molecule and a molecule containing anantibody combining site such as a whole antibody molecule or a portionthereof.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules.Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and portions of animmunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂ and F(v), as well as chimeric antibody molecules.

As used herein, the phrase “immunologically similar to” or“immunologically mimic” refers to the ability of an oligo- orpoly-saccharide of the invention to immunoreact with, or bind to, anantibody of the present invention that recognizes and binds to a nativeantigenic determinant on the capsular polysaccharide of V. choleraeO139.

It should be understood that an oligo- or poly-saccharide of theinvention need not be structurally identical to the capsularpolysaccharide of V. cholerae O139 so long as it is able to elicitantibodies that immunoreact with, or bind to, the capsularpolysaccharide of V. cholerae O139.

An oligo- or poly-saccharide of the invention includes any substitutedanalog, fragment or chemical derivative (either natural or synthetic) ofthe capsular polysaccharide of V. cholerae O139 so long as the oligo- orpoly-saccharide is capable of reacting with antibodies that immunoreactwith the capsular polysaccharide of V. cholerae O139. Therefore, anoligo- or poly-saccharide can be subject to various changes that providefor certain advantages in its use. For example, it has been observedthat loss of the colitose residues from the capsular polysaccharideabolishes antigenicity, and therefore at least one important antigenicdeterminant of V. cholerae O139 CPS comprises or consists of one or morecolitose residues. Synthetic portions of V. cholerae O139 capsularpolysaccharide, and analogs thereof, may be prepared by those skilled inthe art of carbohydrate synthesis [see, e.g., reference 46].

The terms “substitute”, “substituted” and “substitution” include the useof a chemically derivatized residue in place of a non-derivatizedresidue provided that the resulting modified oligo- or poly-saccharidedisplays the requisite immunological activity.

“Chemical derivative” refers to a modified oligo- or poly-saccharidehaving one or more residues chemically derivatized by reaction of afunctional side group. For example, one or more hydroxyl groups of theoligo- or poly-saccharide may be reduced, oxidized, esterified, oretherified; or one or more acetamido groups may be hydrolyzed orreplaced with other carboxamido or ureido groups, and suitably disposedpairs of hydroxyl groups may be converted into cyclic phosphatediesters. Such transformations are well-known and within the abilitiesof those skilled in the art of carbohydrate chemistry. Additionalresidues may also be added for the purpose of providing a “linker” bywhich the modified oligo- or poly-saccharide of this invention can beconveniently affixed to a label or solid matrix or carrier. Suitableresidues for providing linkers may contain amino, carboxyl, orsulfhydryl groups, for example. Labels, solid matrices and carriers thatcan be used with the oligo- or poly-saccharide of this invention aredescribed hereinbelow.

Polymeric Carriers

Carriers are chosen to increase the immunogenicity of the oligo- orpoly-saccharide and/or to raise antibodies against the carrier which aremedically beneficial. Carriers that fulfill these criteria are describedin the art (see, e.g., references 54-59). Polymeric carriers can be anatural or a synthetic material containing one or more primary and/orsecondary amino groups, azido groups, or carboxyl groups. The carriercan be water soluble or insoluble.

Examples of water soluble peptide carriers include, but are not limitedto, natural or synthetic peptides or proteins from bacteria or virus,e.g., tetanus toxin/toxoid, diphtheria toxin/toxoid, Pseudomonasaeruginosa exotoxin/toxoid/protein, pertussis toxin/toxoid, Clostridiumperfringens exotoxins/toxoid, and hepatitis B surface antigen and coreantigen. Mutants of these peptides, derived for example by amino acidsubstitution or deletion, may also be employed as carriers. Toxins,toxoids and mutants of toxins having reduced toxicity are preferredcarriers.

Polysaccharide carriers include, but are not limited to, capsularpolysaccharides from microorganisms such as the Vi capsularpolysaccharide from S. typhi, which contains carboxyl groups and whichis described in U.S. Pat. No. 5,204,098, incorporated by referenceherein; Pneumococcus group 12 (12F and 12A) polysaccharides, whichcontain a terminal galactose: and Haemophilus influenzae type dpolysaccharide, which contains an amino terminal; as well as plant,fruit, or synthetic oligo- or polysaccharides which are immunologicallysimilar to such capsular polysaccharides, such as pectin,D-galacturonan, oligogalacturonate, or polygalacturonate, which aredescribed in U.S. Pat. No. 5,738,855, incorporated by reference herein.

Example of water insoluble carriers include, but are not limited to,aminoalkyl-SEPHAROSE™ (cross-linked agarose), e.g., aminopropyl oraminohexyl SEPHAROSE™ (cross-linked agarose), and aminopropyl glass andthe like. Other carriers may be used when an amino or carboxyl group isadded through covalent linkage with a linker molecule.

Methods for Attaching Polymeric Carriers

The oligo- or poly-saccharides of the invention may be bound to both acarrier saccharide and a non-toxic non-host protein carrier or directlyto a non-toxic non-host protein carrier to form a conjugate.

When the oligo- or poly-saccharide of the invention is bound to both acarrier saccharide and a non-toxic non-host protein carrier, it may bebound first to the carrier saccharide, then the saccharide-carrierconjugate can be bound to the non-toxic non-host protein carrier. Thecomplex compound would properly be described as a semi-synthetic complexmolecule with three distinct domains and origins. This complex compoundwould first contain an oligo- or poly-saccharide bound to the carrierpolysaccharide and then the two-domain saccharide bound to a protein.Alternatively, the oligo- or poly-saccharide of the invention may bebound to both a carrier saccharide and a non-toxic non-host proteincarrier simultaneously.

Methods for binding a polysaccharide to a protein, with or without alinking molecule, are well known in the art. See for example reference[60], where 3 different methods for conjugating Shigella O-SP to tetanustoxoid are exemplified. See also reference [22], which describes methodsfor conjugating S. typhi Vi and adipic hydrazide-derivatized proteins.In U.S. Pat. No. 5,204,098 and U.S. Pat. No. 5,738,855, it is taughtthat an oligo- or poly-saccharide containing at least one carboxylgroup, through carbodiimide condensation, may be thiolated withcystamine, or aminated with adipic dihydrazide, diaminoesters,ethylenediamine and the like. Groups which could be introduced by thismethod, or by other methods known in the art, include thiols,hydrazides, amines and carboxylic acids. Both the thiolated and theaminated intermediates are stable, may be freeze dried, and may bestored at low temperature. The thiolated intermediate may be reduced andcovalently linked to a polymeric carrier containing a sulfhydryl group,such as a 2-pyridyldithio group. The aminated intermediate may becovalently linked to a polymeric carrier containing a carboxyl groupthrough carbodiimide condensation.

The oligo- or poly-saccharide can be covalently bound to a carrier withor without a linking molecule. To conjugate without a linker, forexample, a carboxyl-group-containing oligo- or poly-saccharide and anamino-group-containing carrier are mixed in the presence of a carboxylactivating agent, such as for example a carbodiimide, in a choice ofsolvent appropriate for both the oligo- or poly-saccharide and thecarrier, as is known in the art [58]. The oligo- or poly-saccharide ispreferably conjugated to a carrier using a linking molecule. A linker orcrosslinking agent, as used in the present invention, is preferably asmall linear molecule having a molecular weight of approximately <500and is non-pyrogenic and non-toxic in the final product form (54-59). Toconjugate with a linker or crosslinking agent, either or both of theoligo- or poly-saccharide and the carrier may be covalently bound to alinker first. The linkers or crosslinking agents are homobifunctional orheterobifunctional molecules, e.g., adipic dihydrazide, ethylenediamine, cystamine, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-N-(2-iodoacetyl)-β-alaninate-propionate (SIAP),succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC),3,3′-dithiodipropionic acid, and the like. Dicarboxylic aciddihydrazides are preferred. In the examples presented herein, the linkeris adipic acid dihydrazide, attached via hydrazide linkages to carboxylgroups of the oligosaccharide and the polypeptide. Similar results wouldbe expected with any two- to ten-carbon dihydrazide linker. Otheramino-containing linkers may similarly be bound to carboxyl groups ofthe oligo- or poly-saccharide or the carrier through carbodiimidecondensation. Carboxylic acid containing linkers may be bound to theamino groups of the carrier by means of carboxyl activating reagents(e.g., carbodiimide condensation) or via N-hydroxysuccinimidyl esters orother reactive derivatives. The unbound materials are removed byphysico-chemical methods such as gel filtration or ion exchange columndepending on the materials to be separated. The final conjugate consistsof the oligo- or poly-saccharide and the carrier bound through a linker.

In the present invention, attachment of the V. cholerae capsularpolysaccharide to a protein carrier is preferably accomplished by firstcoupling a dicarboxylic acid dihydrazide linker to the CPS, by treatmentwith a carboxyl activating reagent, such as a water-soluble carbodiimide(e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (DEC) or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC)), butpreferably through one or more hydroxyl groups, using for example1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), to produce ahydrazide-functionalized polysaccharide. Adipic acid dihydrazide is aparticularly preferred linker, but conjugates employing other linkers,such as the dihydrazides of succinic, suberic, and sebacic acids, arecontemplated to be within the scope of the invention. Thelinker-functionalized V. cholerae capsular polysaccharide (CPS_(AH)) isthen coupled to the carrier protein, preferably with a water-solublecarbodiimide, most preferably EDC. In an alternative embodiment, thecarrier protein (rDT) is first coupled to the linker, again using awater-soluble carbodiimide, preferably EDC, and thelinker-functionalized carrier (rDT_(AH)) is then coupled to the CPS witha carboxyl activating reagent, or preferably by hydroxyl coupling usingfor example CDAP. For preparation of the conjugates of this invention,activation of CPS for coupling (with linker or with rDT_(AH)) ispreferably carried out with CDAP, and activation of rDT (for couplingwith linker or with CPS_(AH)) is most preferably carried out with EDC.

Dosage for Vaccination

The present inoculum contains an effective, immunogenic amount of oligo-or poly-saccharide carrier conjugate of this invention. The effectiveamount of oligo- or poly-saccharide carrier conjugate per unit dosesufficient to induce an immune response to V. cholerae, in particular V.cholerae O139, depends, among other things, on the species of mammalinoculated, the body weight of the mammal and the chosen inoculationregimen as is well known in the art. Inocula typically contain oligo- orpoly-saccharide carrier conjugates with concentrations of oligo- orpoly-saccharide of about 1 micrograms to about 100 milligrams perinoculation (dose), preferably about 3 micrograms to about 100micrograms per dose, most preferably about 5 micrograms to about 50micrograms, and most preferably about 5 micrograms to about 25micrograms per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of active material (oligo- orpoly-saccharide conjugate) calculated to produce the desired immunogeniceffect in association with the required diluent.

Inocula are typically prepared as a solution in a physiologicallytolerable (acceptable) diluent such as water, saline orphosphate-buffered saline or other physiologically tolerable diluent toform an aqueous pharmaceutical composition.

The route of inoculation may be intramuscular, subcutaneous and thelike, which results in eliciting antibodies protective against V.cholerae, in particular V. cholerae O139. The dose is administered atleast once. In order to increase the antibody level, a second or boosterdose may be administered approximately 4 to 6 weeks after the initialinjection. Subsequent doses may be administered as indicated.

Adjuvants, such as aluminum hydroxide, QS-21, TITERMAX™ (immunoadjuvant)(CytRx Corp., Norcross Ga.), Freund's complete adjuvant, Freund'sincomplete adjuvant, interleukin-2, thymosin, and the like, may also beincluded in the compositions.

Antibodies

An antibody of the present invention in one embodiment is characterizedas comprising antibody molecules that immunoreact with the capsularpolysaccharide of V. cholerae O139.

An antibody of the present invention is typically produced by immunizinga mammal with an immunogen or vaccine containing a molecular conjugateof the V. cholerae O139 capsular polysaccharide (or a structurallyand/or immunologically related molecule) in an amount sufficient toinduce, in the mammal, antibody molecules having immunospecificity forthe capsular polysaccharide of V. cholerae O139. The capsularpolysaccharide or related molecule is preferably conjugated to acarrier. The antibody molecules may be collected from the mammal andisolated by methods known in the art.

For administration to humans, human or humanized monoclonal antibodiesare preferred, including those made by phage display technology or bynon-human mammals engineered to produce human antibodies.

The antibody molecules of the present invention may be polyclonal ormonoclonal. Monoclonal antibodies may be produced by methods known inthe art. Portions of immunoglobulin molecules, such as Fabs, may also beproduced by methods known in the art.

The antibody of the present invention may be contained in blood plasma,serum, hybridoma supernatants and the like. Alternatively, the antibodyof the present invention is isolated to the extent desired by well knowntechniques such as, for example, ion chromatography or affinitychromatography. The antibodies may be purified so as to obtain specificclasses or subclasses of antibody such as IgM, IgG, IgA, IgG₁, IgG₂,IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferred forpurposes of passive protection.

The antibodies of the present invention have a number of diagnostic andtherapeutic uses. The antibodies can be used as an in vitro diagnosticagent to test for the presence of V. cholerae, in particular V. choleraeO139, in biological samples in standard immunoassay protocols. Suchassays include, but are not limited to, agglutination assays,radioimmunoassays, enzyme-linked immunosorbent assays, fluorescenceassays, Western blots and the like. In one such assay, for example, thebiological sample is contacted to antibodies of the present inventionand a labeled second antibody is used to detect the presence of V.cholerae, in particular V. cholerae O139, or the capsular polysaccharideantigen of V. cholerae, in particular V. cholerae O139, to which theantibodies are bound.

Such assays may be, for example, of direct format (where the labeledfirst antibody is reactive with the antigen), an indirect format (wherea labeled second antibody is reactive with the first antibody), acompetitive format (such as the addition of a labeled antigen), or asandwich format (where both labeled and unlabelled antibody areutilized), as well as other formats described in the art.

The antibodies of the present invention are useful in prevention andtreatment of infections and diseases caused by V. cholerae, inparticular V. cholerae O139.

In providing the antibodies of the present invention to a recipientmammal, preferably a human, the dosage of administered antibodies willvary depending upon such factors as the mammal's age, weight, height,sex, general medical condition, previous medical history and the like.

In general, it is desirable to provide the recipient with a dosage ofantibodies which is in the range of from about 1 mg/kg to about 10 mg/kgbody weight of the mammal, although a lower or higher dose may beadministered.

The antibodies of the present invention are intended to be provided tothe recipient subject in an amount sufficient to prevent, lessen orattenuate the severity, extent or duration of the infection by V.cholerae, in particular V. cholerae O139. Antibodies which immunoreactwith DT may also be provided to a recipient subject in an amountsufficient to prevent, lessen or attenuate the severity, extent orduration of an infection by Corynebacterium diptheriae.

The administration of the agents of the invention may be for either“prophylactic” or “therapeutic” purpose. When provided prophylactically,the agents are provided in advance of any symptom. The prophylacticadministration of the agent serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection. Theagent of the present invention may, thus, be provided either prior tothe anticipated exposure to V. cholerae, in particular V. cholerae O139,(so as to attenuate the anticipated severity, duration or extent of aninfection and disease symptoms) or after the initiation of theinfection.

For all therapeutic, prophylactic and diagnostic uses, the oligo- orpoly-saccharide of the invention, alone or linked to a carrier, as wellas antibodies and other necessary reagents and appropriate devices andaccessories may be provided in kit form so as to be readily availableand easily used.

The following examples illustrate certain embodiments of the presentinvention, but should not be construed as limiting its scope in any way.Certain modifications and variations will be apparent to those skilledin the art from the teachings of the foregoing disclosure and thefollowing examples, and these are intended to be encompassed by thespirit and scope of the invention.

EXAMPLES

The examples describe two methods for the synthesis of a conjugatecomprising the capsular polysaccharide of V. cholerae O139, with ahomobifunctional linker unit used for covalent attachment to a mutantdiphtheria toxin as a model carrier protein. These examples are alsodescribed in reference 47.

Materials and Methods

Materials. Chicken serum albumin Fraction V (CSA), rabbit CSA antiserum,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), adipic aciddihydrazide (ADH), 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP), and agarose were from Sigma Chemical Co., St Louis, Mo.;SEPHAROSE™ (cross-linked agarose) CL-4B and SEPHADEX™ (cross-linkeddextran) G-25 from Pharmacia AB, Uppsala, Sweden; BSA standard solution,Coomassie blue protein assay reagent, triethylamine (TEA) from Pierce,Rockford, Ill., nickel nitrilotriacetic acid (NiNTA) chelating agarosefrom Qiagen Inc., Chatsworth, Calif.; acetonitrile from T. J. Baker,Inc., Philipsburg, N.J.; diphtheria toxin (DT) from List BiologicalLaboratories, Inc, Campbell, Calif., equine antidiphtheria toxin,Lederle Laboratories, Pearl River N.Y., Lot 152-5456 R a gift from CBER,FDA; rabbit (3-4 week) complement from Pel-Freez, Brown Deer, Wis.;dialysis membranes (molecular weight cut off 6-8,000) from Spectra-Por,Laguna Hills, Calif.; ultrafiltration membrane YM100 and CENTRIPREP™(cellulose membrane) 30 from Amicon, Inc, Beverly, Mass.; Limulusamebocyte lysate pyrogen (U.S. License No. 709) from BioWhittaker, Inc.,Walkersville, Md.; tryptic soy broth (TSB) from Difco Inc, Detroit,Mich. (TSB containing 1% agarose was denoted as TSA). Deionized orpyrogen-free water (PFW) and pyrogen-free saline (PFS) were used in allexperiments.

Bacteria. V. cholerae O139 MDO-12C [8], a heavily capsulated and opaquevariant selected from the isolate MDO-12 (Madurai, India), was used forpreparation of CPS and murine hyperimmune serum. V. cholerae O139SPH1168, a clinical isolate from a Thai patient (Suanphung Hospital,Thailand), was used as the target strain in the vibriocidal assay. Bothisolates were stored in 20% glycerol at −70° C.

Purification of V. cholerae O139 CPS. V. cholerae O139 MDO-12C waspropagated from a single colony on TSA to 4×100 ml and then to 4×1 L ofTSB for 5 h at 37° C. with shaking at 200 rpm. The 4-L inoculum(A₅₆₀˜3.0) was transferred to a 300-L fermenter containing 150 L of TSB,0.1% dextrose and 0.05 M MgSO₄. Fermentation was conducted at 30%dissolved oxygen, 35° C. and pH 7.0 (maintained with NH₄OH). After 16 h,formalin was added to a final concentration of 2% and stirred slowly for6 h at room temperature. The suspension was centrifuged and thesupernatant concentrated to 1.2 L by ultrafiltration and stored at −20°C.

A 500-mL aliquot of the concentrated supernatant was mixed with 3volumes of 95% ethanol and stored overnight at 4° C. The supernatant wasdecanted and the slurry spun down at 10,500×g, 10° C. for 30 min. Thepellet (20 g wet weight) was washed with 80% ethanol, dissolved in 800ml of 10% saturated sodium acetate, pH 7.5, and extracted with coldphenol 3 times [9]. The final water phase was dialyzed against H₂O for 3days at 4-8° C. and freeze-dried. The precipitate was dissolved in 150ml of 0.1 M CaCl₂ and ultracentrifuged at 145,000×g, 10° C. for 5 h. Thesupernatant was recentrifuged as above, dialyzed against H₂O,freeze-dried (yield 1.6 g) and stored at −20° C.

This material (unfractionated CPS) was dissolved in PFW (100 mg/50 ml)and passed through an Amicon membrane YM100. The retentate was passedthrough a 2.5×90 cm column of SEPHAROSE™ (cross-linked agarose) CL-4B inPFS. The retentate was eluted from the column as one peak at Kd 0.4.Colitose-containing fractions were pooled, dialyzed against PFW andfreeze-dried. This material was denoted as CPS and used to prepareconjugates with rDT. In earlier experiments the filtration through theAmicon membrane was omitted (see preparation of CPS-AH conjugatesbelow).

¹³C NMR spectroscopy. ¹³C NMR spectrum of the CPS (50 mg/ml D₂O) wasmeasured using Varian XL3000 spectrometer by averaging 50,000 scans witha 10-s decay between acquisition and 10-μs 90° pulse. Prior to Fouriertransformation, a 5-Hz line broadening was applied and zero-filled to32,000 datum points.

Murine hyperimmune V. cholerae O139 serum. V. cholerae O139 culture wasprepared by transferring a single colony from TSA to 50 ml of LB andincubating at 37° C., 200 rpm for 5 h (A₅₆₀˜1.0). The culture wasinactivated with 1% formalin. Thirty 6-week-old female Swiss mice (NIH)were injected as follows: 1) 3 subcutaneous injections of 100 μL 1 dayapart; 2) after 9 days, 3 intraperitoneal injections of 150 μL 1 dayapart; 3) 9 days later, 3 intravenous injections of 200 μL 1 day apart.Mice were exsanguinated seven days after the last injection. All serashowed a precipitin line by double immunodiffusion with CPS: a pool wasdenoted as murine hyperimmune V. cholerae O139 serum.

Purification of recombinant diphtheria toxin mutant CRMH21G (rDT). TherDT was constructed by site-directed mutagenesis on the A chainreplacing histidine at position 21 with glycine and expressed inEscherichia coli BL21 (λDE3) [15]. To facilitate purification by NiNTA,a 6-histidine tag was attached to the protein carboxyl terminal.Fermentation of this recombinant strain was performed as described [5].The cell paste was suspended in 0.5 M NaCl, 0.02 M Tris, 0.005 Mimidazole, pH 8.0 and the cytoplasm was released by a French press. Thesupernatant was passed through a 2.5×10 cm column NiNTA and washed with0.02 M Tris buffer containing 0.03 M imidazole (pH 8.0). rDT was elutedwith 0.02 M Tris buffer containing 0.25 M imidazole, pH 8.0 at 3-8° C.The eluate was dialyzed exhaustively against 0.02 M Tris, pH 8.0 at 3-8°C. (yield 150 mg rDT/L supernatant). Prior to derivatization, rDT wasdialyzed at 3-8° C. against PBS with multiple changes of outer fluidfollowed by dialysis against 0.2 M NaCl, pH 7.2-7.7 (adjusted with 1 MNaOH). The protein solution was concentrated to ˜10 mg/ml in an AmiconCENTRIPREP™ (cellulose membrane) 30.

rDT had the same R_(f) in 10% SDS-PAGE and the same circular dichroismspectrum as DT. A line of identity was formed between rDT and DT whenreacted against equine anti-DT by double immunodiffusion.

Adipic acid hydrazide (AH) derivatives. CPS was treated with ADH in thepresence of CDAP [20, 21, 23], while CSA and rDT were treated with ADHin the presence of EDC [22, 37] as the activating agent.

AH derivative of CSA (CSA_(AH)). The AH derivative of CSA (CSA_(AH)) wasprepared by EDC-mediated condensation of ADH and CSA [18].Concentrations of the reactants in the reaction mixture were 10 mgCSA/mL, 0.2 M ADH, and 0.015 M EDC. ADH (powder) was added to the CSA,the pH adjusted to 5.5 with 0.1 M MES buffer (pH 5.5), and EDC (powder)was added. The reaction was carried out at room temperature, pH 5.5 to5.7 for 1 h. The mixture was dialyzed overnight at 4° C. against salineand passed through a 2.5×40 cm column of SEPHADEX™ (cross-linkeddextran) G-25 in saline. The void volume fractions were pooled,concentrated by ultrafiltration, stored at 4° C., and designated asCSA_(AH).

AH derivatives of CPS

CPS_(AH). CDAP activation of CPS was performed as described [21] using aCDAP:CPS ratio of 1:5 (w/w). 40 mg of CPS (not filtered through an theAmicon YM100 membrane) was dissolved in 1.5 ml of H₂O; pH 5.0. 80 μL ofCDAP in acetonitrile (100 mg/ml) were added with stirring followed in 30sec with 80 μL of 0.2 M TEA. After 2 min, the pH was adjusted to 7.8with 0.1 MHCl. Then 1.5 ml of 0.8 M ADH in 0.5 M NaHCO₃ was added andthe pH maintained between 8.0 and 8.4 with 0.1 M HCl for 2 h at roomtemperature. The reaction mixture was dialyzed overnight against 6 L ofwater with 2 changes, and passed through a 2.5×40 cm column of SEPHADEX™(cross-linked dextran) G-25 in PFW. The void volume fractions werefreeze-dried, denoted as CPS_(AH), and assayed for AH.

CPS_(AH1). CPS which had been filtered through the Amicon YM100 membranewas employed. Concentrations of the reactants in the reaction mixturewere 20 mg/mL of CPS, 0.05M ADH, and 0.05 M EDC. MES buffer (pH 5.5) wasadded to the CPS in water to adjust the pH to 5.5. EDC and ADH (both inpowder form) were then added. The reaction was carried out for 2 h atroom temperature and pH 5.5-5.6 was maintained with 0.5 M MES-acid. ThepH was then brought to 7.0 with 0.1 M sodium phosphate buffer (pH 8.0),dialyzed overnight against water, and passed through a 1.5×24 cm columnof Bio-gel P-10 in water. The void volume fractions were freeze-driedand designated as CPS_(AH1).

CPS_(AH2). CPS which had been filtered through the Amicon YM100 membranewas employed. The reaction was performed as described [21], at aCDAP/CPS ratio of 3:10 (w/w). 1 mL of CPS (30 mg/mL water), pH 5.0, wasmixed with 90 μL of CDAP in acetonitrile (100 mg/mL). After 30 sec, 90μL of 0.2 M TEA was added. During the next 2 min the pH dropped from 8.1to 7.2, and 1 mL of 0.8 M ADH in 0.5 M NaHCO3 was added. The reactionwas carried out for 2 h at room temperature and a pH 8.3-8.6 maintainedwith 0.1 M NaOH. The mixture was dialyzed overnight against water andpassed through a 2.5×40 cm column of SEPHADEX™ (cross-linked dextran)G-25 in water. The void volume fractions were freeze-dried and denotedas CPS_(AH2).

AH derivative of rDT (rDT_(AH)). The reaction mixture contained 10 mg/mlof protein, 0.2 M ADH and 0.011 M EDC. The reaction was carried out for1 h at room temperature with the pH maintained at 6.2-6.4 with 0.1 MHCl. The reaction mixture was dialyzed against 0.2 M NaCl, pH 7.0(adjusted with 1 M NaOH), and passed through a 1.5×25 cm column ofSEPHADEX™ (cross-linked dextran) G-25 in the same solution. Void volumefractions were pooled, concentrated (Amicon CENTRIPREP™ (cellulosemembrane) 30), denoted as rDT_(AH), and assayed for protein and AH.

Conjugates with CSA. Two sets of conjugates, CPS-CSA_(AH) andCPS_(AH)-CSA were prepared using EDC and CDAP as the activating agents[21, 22].

EDC-mediated synthesis of CPS-CSA_(AH). The concentration of reactantsin the reaction mixture were 10 mg/mL of CPS and 10 mg/mL of CSA_(AH),and 0.02 M EDC. CPS was mixed with CSA_(AH), and the pH was adjusted to5.5 with 0.5 M MES buffer (pH 5.5). The mixture was brought to the finalvolume with saline, and EDC was added as powder. The reaction wascarried out at room temperature for 3 h, during which the pH rose from5.5 to 5.7. The conjugation was accompanied by a formation ofprecipitate that became gradually heavier. The mixture was dialyzedovernight against saline and centrifuged (7,000×g, 5 min) before passingthrough a 1.5×90 cm column of SEPHAROSE™ (cross-linked agarose) CL-2B insaline. Fractions were assayed for polysaccharide and protein. Thefractions 21-29 of Vo peak were pooled and denoted as EDC:CPS-CSA_(AH).

CDAP-mediated synthesis of CPS-CSA_(AH). CPS was activated with CDAP andbound to CSA_(AH) at a CDAP/CPS ratio of 3:10 (w/w). 10 mg of CPS inwater (100 mg/mL) was mixed with 30 μL of CDAP in acetonitrile (100mg/mL). The mixture (pH 5.2) was stirred for 30 sec, and 30 μL of 0.2 MTEA was added. After 2 min, 0.1 M NaOH was added to bring the pH from7.0 to 8.2. CSA_(AH) (10 mg) was added, and the volume adjusted withsaline to 2 mL. The reaction was carried out for 3 h at roomtemperature, and a pH of 8.0 to 8.3 was maintained with 0.1 M NaOH. Themixture was passed through a 1.5×90 cm column of SEPHAROSE™(cross-linked agarose) CL-2B in saline. Fractions were assayed forpolysaccharide and protein. Fractions 30 to 46 were pooled and denotedas CDAP:CPS-CSA_(AH).

EDC-mediated synthesis of CPS_(AH1)-CSA and CPS_(AH2)-CSA.

CPS_(AH1)-CSA. Concentrations of the reactants in the reaction mixturewere 10 mg/mL of CPS_(AH)1, 10 mg/mL of CSA, and 0.02 M EDC. CPS_(AH1)was mixed with CSA, and the pH was adjusted to 5.5 with 0.5 M MES buffer(pH 5.5). EDC was added as powder, and the mixture was brought to thefinal volume with saline. The reaction was carried out at roomtemperature for 3 h during which the pH rose from 5.5 to 5.6. Thereaction mixture was passed through a 1.5×90 cm column of SEPHAROSE™(cross-linked agarose) CL-2B in saline. Fractions were assayed forpolysaccharide and protein. Fractions 36 to 52 were pooled and denotedas EDC:CPS_(AH1)-CSA.

CPS_(AH2)-CSA. Concentrations of the reactants in the reaction mixturewere 5 mg/mL of CPS_(AH2), 5 mg/mL of CSA, and 0.05 M EDC. The procedurewas performed as described above. Fractions were assayed forpolysaccharide and protein. Fractions 36 to 52 were pooled and denotedas EDC:CPS_(AH2)-CSA.

Conjugates with rDT. Two schemes were used to prepare conjugates withrDT: 1) EDC-mediated conjugation of the CPS_(AH) with rDT, and 2)CDAP-mediated conjugation of the CPS with rDT_(AH).

EDC-mediated conjugation of CPS_(AH) with rDT. Each reaction mixturecontained 8 mg/ml of CPS_(AH) and of rDT, and EDC of 0.05 M (forI:CPS_(AH)-rDT) or 0.02 M (for II:CPS_(AH)-rDT).

CPS_(AH) was dissolved in 0.2 M NaCl and the pH adjusted to 6.2 with 0.1M NaOH. rDT was added and the volume adjusted with 0.2 M NaCl. Afterstirring for 1 min, EDC was added. The reaction was carried out for 3 hat room temperature and the pH maintained at 6.2-6.4 with 0.1 M HCl. Themixture was dialyzed overnight at 3-8° C. against 0.2 M NaCl, 0.005 Msodium phosphate, pH 7.5, and passed through a 1.5×90 cm column ofSEPHAROSE™ (cross-linked agarose) CL-4B in the same buffer. Fractionswere assayed for polysaccharide and protein. The void volume fractionswere pooled and denoted as I:CPS_(AH)-rDT and II:CPS_(AH)-rDT.

CDAP-mediated conjugation of CPS with rDT_(AH). Each reaction mixturecontained 8 mg/ml of CPS and of rDT_(AH): the CDAP/CPS was 4:5 (forI:CPS-rDT_(AH)) or 1:5 (for II:CPS-rDT_(AH)).

CDAP (100 mg/ml acetonitrile) was added to CPS in 0.2 M NaCl (pH 5.2)and mixed for 30 sec. An equal volume of 0.2 M TEA to that of CDAP wasadded. After 2 min, the pH dropped from 8.5 to 7.2 and rDT_(AH) wasadded. The pH was raised from 7.2 to 8.3 with 0.1 M NaOH. The reactionwas carried out for 2 h at room temperature during which the pH wasstable. The mixture was dialyzed overnight against 0.2 M NaCl, 0.005 Msodium phosphate buffer, pH 7.5, and passed through a 1.5×90 cm columnof SEPHAROSE™ (cross-linked agarose) CL-4B in the same buffer. Fractionswere assayed for polysaccharide and protein and void volume fractionspooled and denoted as I:CPS-rDT_(AH) and II:CPS-rDT_(AH).

Chemical assays. Polysaccharide was assayed by measuring3,6-dideoxyhexose (colitose) with the CPS as the standard [18]. Proteinwas measured by Coomassie blue assay with BSA as the standard [2].Hydrazide content of CPS_(AH) and rDT_(AH) was measured by the TNBSmethod using ADH as the standard [14]. The degree of derivatization wasexpressed in % of AH, and the mol/mol ratio of AH to polysaccharide orto protein.

Limulus amebocyte lysate test. CPS was assayed for endotoxin by limulusamebocyte lysate test. The FDA Reference Standard Endotoxin (Lot EC-5)was used as a reference for the assay. The test conforms with the FDAguideline [41].

Immunodiffusion. Double immunodiffusion of the conjugates was performedin 1% agarose gel in 0.15 M NaCl with murine hyperimmune cholera O139serum and equine diphtheria toxin antiserum.

Immunization of mice. Six-week-old female Swiss albino mice (10 pergroup) were injected subcutaneously 3 times at 2-week intervals with 100μL of immunogen containing 2.5 μg of the CPS alone or as the conjugate.A control group received 1 injection of 100 μL of saline. Mice wereexsanguinated 7 days after each injection and sera stored at −20° C.

ELISA. Flat-bottom 96-well microtiter plates (NUNC-IMMUNO™ (coatedpolystyrene), Denmark) were coated with CPS (20 μg/ml PBS) and keptovernight at room temperature. After washing with 0.15 M NaCl, 0.1% Brijand 3 mM sodium azide, plates were blocked with 1% BSA in PBS for 2 h atroom temperature. The plates were washed and 2-fold serial dilutions ofsera in 1% BSA, 0.1% Brij, PBS added. Reference serum was assayed intriplicates and samples in duplicates. Plates were incubated overnightat room temperature, washed, and the alkaline phosphatase-labeled goatantibody specific to mouse IgG or for IgM was added. After 4 h at roomtemperature, the plates were washed, and the 4-nitrophenyl phosphatesubstrate (1 mg/ml in 1 M Tris-HCl, 3 mM MgCl₂, pH 9.8) was added. A₄₀₅was measured by a MRX Dynatech reader.

Anti-CPS IgG was measured in all murine sera; anti-CPS IgM was measuredonly in 11 representative sera from mice injected 3 times withII:CPS_(AH)-rDT or I:CPS-rDT_(AH). Murine hyperimmune V. cholerae O139serum was used as the reference for both anti-CPS IgG and IgM. Thisserum was arbitrarily assigned a value of 1000 ELISA units/ml (EU) forIgG and 100 EU for IgM upon the observation that 1/20,000 dilution ofanti-IgG and 1/100 dilution of anti-IgM gave approximately the sameA₄₀₅.

An analogous ELISA procedure was used to measure anti-DT IgG: plateswere coated with DT (5 μg/ml) and a mouse serum with high titer ofanti-DT IgG, arbitrarily assigned a value of 1000 EU, served as thereference.

ELISA results were computed with an ELISA Data Processing Programprovided by the Biostatistics and Information Management Branch, CDCbased upon four parameters logistic-log function using Taylor SeriesLinearization Algorithm [34]. Anti-CPS IgG and anti-DT IgG levels areexpressed as geometric means.

Statistics. Comparisons of the geometric means were performed with thetwo-sided t test or Wilcoxon analysis.

Vibriocidal assay. Eleven representative sera from mice injected threetimes with II:CPS_(AH)-rDT or I:CPS-rDT_(AH), and twenty convalescentsera from cholera patients infected with V. cholerae O139 (SamutskakornHospital, Thailand) [13] were assayed for vibriocidal activity beforeand following treatment with 0.1 M 2-ME for 30 min at 37° C. [10, 27].The patient sera were also tested for fibriocidal activity afterabsorption with CPS in vibriocidal antibody inhibition assay (VAI) [6].

Bacteria were prepared by transferring a single colony from TSA into 10ml of TSB and incubating for 2 to 3 h at 37° C. with shaking at 180 rpm.100 μL of this inoculum was transferred to 10 ml of TSB and incubatedwith shaking (180 rpm) at 37° C. until culture reached A₅₆₀ of 0.2-0.24(3.0-4.0×10⁷ cells/ml). The bacterial suspension was diluted 10⁵-fold inDulbecco's buffer.

Vibriocidal assay was performed in sterile non-pyrogenic 24-well cellculture plates (Costar, Corning, N.Y.) by mixing equal volumes of serum,bacteria and complement. The tested serum was 2-fold serially diluted inDulbecco's buffer (for VAI in 100 mg CPS/ml Dulbecco's buffer), so thateach well contained 100 μL. 100 μL aliquots of the bacteria and ofcomplement were added into each well. Plates were incubated for 1 h at37° C. with shaking. Two 100 μL aliquots from each well were transferredinto empty wells and 1 ml of TSA (46-48° C.) added to all 3 wells.Plates were incubated overnight at 37° C. and the colonies counted. Thevibriocidal titer was defined as the reciprocal of the highest serumdilution showing ≧60% reduction in number of colonies compared to thecontrol (complement only) [25].

Results

V. cholerae O139 CPS. The V. cholerae O139 CPS isolated from culturesupernatant (Material and Methods) showed three peaks at Kds of 0.4,0.71 and 0.91 on SEPHAROSE™ (cross-linked agarose) CL-4B with yields of70%, 29% and 1%, respectively (FIG. 1). Colitose, a component of theCPS-repeating unit, was detected only in the peak at Kd 0.4. Fastseparation of this peak-material from the lower molecular weightmaterials (Kds 0.71 and 0.91) was accomplished by diafiltration of theunfractionated CPS through an Amicon membrane YM100. To confirm itspurity, the retentate was passed through SEPHAROSE™ (cross-linkedagarose) CL-4B and showed only a peak of Kd 0.4. ¹³C-NMR spectrum of theretentate (equivalent to the peak-material of Kd 0.4) [FIG. 2] wasidentical to a published ¹³C NMR spectrum of V. cholerae O139 CPS [19,35]. The filtrate spectrum, in contrast, lacked chemical shifts forcolitose, quinovosamine, GluNAc and D-galacturonic acid. The retentategave strong reaction with the murine V. cholerae O139 hyperimmune serumby Western blot and double immunodiffusion. The retentate, denoted asthe CPS, showed only <0.5 endotoxin units/μg as measured by limulusamebocyte lysate test. Fractions not containing colitose were notantigenic.

AH derivatives of CSA. CSA_(AH) contained ˜9 moles of AH per mole CSA.CSA_(AH) formed a line of identity with CSA when reacted with rabbitanti-CSA serum by double-immunodiffusion.

AH derivatives of CPS (CPS_(AH), CPS_(AH1), CPS_(AH2),) and rDT(rDT_(AH)) [Table 1]. CPS_(AH1), prepared by EDC-mediated reaction,contained 0.08 moles of hydrazide per mole of CPS-repeating unit.CPS_(AH2), prepared by the CDAP method, contained 0.12 moles ofhydrazide per mole of CPS repeating unit. CPS_(AH) contained 3.4% of AH,which represents ˜1 AH per 5 CPS-repeating units. All three AHderivatives formed a line of identity with CPS when reacted with murinehyperimmune V. cholerae O139 serum by double immunodiffusion.

rDT_(AH) contained 7.2 moles of AH per mole of protein and formed a lineof identity with rDT when reacted with equine DT antiserum by doubleimmunodiffusion.

TABLE 1 Adipic acid hydrazide derivatives (AH) of Vibrio cholerae O139capsular polysaccharide (CPS) and of recombinant diphtheria toxin mutant(rDT) Activating AH content Derivative agent % mol/mol* CPS_(AH) CDAP3.44 0.21 rDT_(AH) EDC 1.90 7.12 *CPS-repeating unit (M_(r) 1053), rDT(M_(r) ~ 67,000)

Conjugates. (FIG. 3, Table 2).

EDC-mediated synthesis of CPS_(AH)-CSA conjugates (EDC:CPS-CSA_(AH)).Gel filtration profile of this conjugate, prepared by EDC-mediatedbinding of CPS to CSA_(AH), showed 2 peaks (Vo and Kd 0.52) thatcontained both polysaccharide (PS) and proptein (PR). The Vo materialconsisted mostly of PR (PS/PR 0.15). The majority of PS was detected inthe second peak (Kd 0.52). Formation of this conjugate was accompaniedby the development of a protein precipitate that accounted for about 30%of total PR. Only Vo material was included in the final pool of theconjugate, and the yield (by the recovery of PS) was 2.7%.

CDAP-mediated synthesis of CPS_(AH)-CSA conjugates (CDAP:CPS-CSA_(AH)).This conjugate was prepared from the same components as EDC:CPS-CSA_(AH)but using CDAP as the activating agent. Gel filtration of this conjugateshowed that PS was present in fractions 30-65, similar to the elutionrange of CPS alone (34-60). PR was detected within Fr 30-70, that is 20fractions before the elution range of CSA alone. Only fractions 30-47 ofthe PS and PR overlapping region were included in the final pool of theconjugate. The PS/PR ratio of the conjugate was 2.6, and the yield basedon the recovery of PS was 51%.

EDC-mediated synthesis of CPS_(AH)-CSA conjugates (EDC:CPS_(AH1)-CSA andEDC:CPS_(AH2)-CSA). Both conjugates were prepared by EDC-mediatedconjugation of the AH derivative of CPS with CSA, but the w/w ratio ofEDC/PR was 5-fold higher in the synthesis of EDC:CPS_(AH2)-CSA. Itshould be also noted that CPS_(AH1) and CPS_(AH2) had a differentcontent of hydrazide and were prepared by different derivatizationreactions. Gel filtration of the conjugates showed that PS and PR peaksoverlapped in the range of 36-52 (EDC:CPS_(AH1)-CSA) and 36-60(EDC:CPS_(AH2)-CSA). There was more PR eluted at Kd 0.78 (identical toKd of CSA alone) in the first conjugate. Only fractions 36-52 wereincluded in the final pools of each conjugate. The PS/PR (w/w) ratio andyield, by the recovery of PS, were higher for EDC:CPS_(AH1)-CSA than forEDC:CPS_(AH2)-CSA.

EDC-mediated synthesis of CPS_(AH)-rDT conjugates. Identical reactionconditions, except for the concentration of EDC, were used to prepareI:CPS_(AH)-rDT (0.05 M EDC) and II:CPS_(AH)-rDT (0.02 M EDC). gelfiltration of either conjugate on SEPHAROSE™ (cross-linked agarose)CL-4B yielded 3 peaks at Vo, Kd 0.4 and Kd 0.76 (FIG. 3A). Two peaks, atVo and Kd 0.4, consisted of both polysaccharide and protein, while thepeak at Kd 0.76 contained only protein. Since the Kds of the free CPSand rDT on SEPHAROSE™ (cross-linked agarose) CL-4B are 0.4 and 0.76,respectively, the presence of unreacted CPS and/or rDT within the rangeof Kd 0.4-0.76 could not be excluded. Accordingly, only the void volumefractions were pooled and denoted as I:CPS_(AH)-rDT and II:CPS_(AH)-rDT.

I:CPS_(AH)-rDT had a lower polysaccharide/protein ratio (w/w) thanII:CPS_(AH)-rDT (0.46<0.76). The yields of both conjugates were about20% based upon the recovery of polysaccharide. Double immunodiffusion ofeither conjugate against murine V. cholerae O139 and equine DT toxinhyperimmune sera showed a single precipitin line (FIG. 4A).

CDAP-mediated synthesis of CPS-rDT_(AH) conjugates. I:CPS-rDT_(AH) andII:CPS-rDT_(AH) were prepared under the same conditions except for thew/w ratio of CDAP/CPS that was 4:5 and 1:5, respectively. Gel filtrationof either conjugate on SEPHAROSE™ (cross-linked agarose) CL-4B showed 2peaks (Vo and Kd 0.4) both containing polysaccharide and protein (FIG.3B). The void volume fractions were pooled and denoted as conjugatesI:CPS-rDT_(AH) and II:CPS-rDT_(AH): their polysaccharide/protein ratioswere 0.99 and 0.90, respectively. The yield of I:CPSrDT_(AH) was 45%.The yield of II:CPS-rDT_(AH) could not be determined because of anaccidental loss of some material. Both conjugates formed a singleprecipitin line when reacted with murine V. cholerae O139 and equine DThyperimmune sera by double immunodiffusion (FIG. 4B).

The structural differences between CPS-rDT_(AH) and CPS_(AH)-rDT areunknown, but differing points of attachment and differing levels ofcrosslinking seem likely.

TABLE 2 Composition of V. cholerae O139 capsular polysaccharide (CPS)conjugates with recombinant diphtheria toxin mutant (rDT). ConjugationPS/PR Conjugate Method (w/w) Yield* I:CPS_(AH)-rDT EDC (0.05 M) 0.4628.0% II:CPS_(AH)-rDT EDC (0.02 M) 0.78 20.1% I:CPS-rDT_(AH) CDAP:CPS(4:5) 0.90 45.0% II:CPS-rDT_(AH) CDAP:CPS (1:5) 0.99 ** *Yielddetermined by the amount of polysaccharide in conjugate **Accidentalloss of some material prevented accurate determination

Serum antibody responses elicited by conjugates (Table 3).

Anti-CPS IgG: All conjugates elicited a significant rise after thesecond injection (P<0.006). Among the rDT conjugates, onlyII:CPS_(AH)-rDT, I:CPS-rDT_(AH) and II:CPS-rDT_(AH) elicited a boosterafter third injection (P<0.003).

Anti-DT IgG: All rDT conjugates elicited significant rises after the 2ndand 3rd injections. After the third injection, II:CPS_(AH)-rDT elicitedthe highest and statistically significantly different level compared toother conjugates (P<0.0007).

Anti-CPS IgG. CPS alone did not elicit an antibody response compared tosaline (0.22 vs. 0.19, NS).

None of the rDT conjugates elicited a statistically significant antibodyresponse after the first dose. All four conjugates elicited significantrises of anti-CPS IgG after the second dose (P<0.006). However, onlyII:CPS_(AH)-rDT, I:CPS-rDT_(AH) and II:CPS-rDT_(AH), elicited a boosterresponse following the third dose compared to the second one (P<0.003).There were no significant differences between the post-third levelselicited by these three conjugates (10.3 vs. 11.5 vs. 4.21 NS): all weresignificantly higher than those elicited by I:CPS_(AH)-rDT (10.3, 11.5,4.21 vs. 0.43, P<0.0001).

Anti-diphtheria toxin IgG. All conjugates elicited significant rises ofanti-DT IgG after the second and third injections compared to the firstinjection (P<0.0001). II:CPS_(AH)-rDT induced the highest level ofanti-DT IgG of all conjugates, however, only the post-third injectionlevel was statistically significantly higher (1050 vs. 245, 255, 279,P<0.0007).

TABLE 3 Serum IgG response specific to CPS and DT elicited in mice (n =10/group) by V. cholerae O139 CPS conjugates with rDT ELISA units/ml(25-75 centiles) Conjugate Dose anti-cps IgG anti-DT IgG I:CPS_(AH)-rDT1 0.13 (0.11-0.17) 0.05 (0.03-0.06) 2 0.35 (0.24-0.5) 52.8 (33.8-87.4) 30.43 (0.28-0.58) 254. (121-376) II:CPS_(AH)-rDT 1 0.23 (0.16-0.28) 0.60(0.2-2.6) 2 1.04 (0.35-0.68) 186. (155-233) 3 10.3 (1.67-12.2) 1050.(715-1210) I:CPS-rDT_(AH) 1 0.11 (0.11-0.14) 0.70 (0.37-1.3) 2 0.89(0.39-1.14) 81.1 (52.3-128) 3 11.5 (4.99-29.7) 255. (128-548)II:CPS-rDT_(AH) 1 0.22 (0.18-0.27) 0.80 (0.34-2.8) 2 0.49 (0.35-0.68)146. (90-214) 3 4.21 (1.67-12.2) 279. (175-388) CPS alone after 3injections did not elicit anti-CPS IgG as compared to saline (0.21 EU vs0.19 EU, NS).

Vibriocidal activity of murine sera (Table 4). Representative sera frommice injected 3 times with CPS conjugates were tested for vibriocidalactivity. The CPS-rDT conjugate induced titers ranged from 1600-6400:II:CPS_(AH)-rDT induced slightly higher titers (3200-6400) thanI:CPS-rDT_(AH) (1600-3200). These two groups of sera showed a similarrange of anti-CPS IgG levels, while the anti-CPS IgM levels wereslightly higher in mice injected with II:CPS_(AH)-rDT than withI:CPS-rDT_(AH). Similar vibriocidal results were demonstrated with theheavily capsulated V. cholerae O139 MDO12C variant (the strain which wasused for purification of the CPS) and other clinical isolates as thetarget strains.

Following treatment with 2-ME, the vibriocidal titers of most seradeclined about 4-fold, however, all retained significant levels ofvibriocidal activity.

TABLE 4 Vibriocidal activity of representative sera from mice injected 3times with the conjugates of V. cholerae O139 capsular polysaccharide(CPS) and chicken serum albumin (CSA) or recombinant diphtheria toxinmutant (rDT). vibriacidal titer anti-CPS (EU) untreated treatedConjugate IgG IgM serum with 2-ME CDAP:CPS-CSA_(AH) 18.4 1.68 1000 —73.9 1.67 2000 — 102.3 1.50 1000 — 325.0 11.53 8000 — EDC:CPS_(AH1)-CSA14.5 2.51 2000 — 58.9 2.38 4000 — 71.9 0.91 2000 — 118.1 2.50 2000 —EDC:CPS_(AH)-CSA 18.1 2.38 1000 — 62.4 2.21 2000 — 70.3 2.32 4000 —103.5 9.92 4000 — II:CPS_(AH)-rDT 13.2 5.43 3200 400 17.5 3.64 1600 40042.2 6.94 3200 800 54.5 9.11 6400 1600 180.8 10.5 >6400 1600I:CPS_(AH)-rDT 15.5 4.02 1600 400 18.9 1.45 1600 800 27.2 2.05 1600 20029.7 2.38 1600 400 36.3 2.54 3200 800 68.5 2.32 3200 800 Serum anti-CPSIgG and IgM levels are expressed in ELISA units/ml (EU) compared to amurine hyperimmune cholera O139 serum arbitiarily assigned 1000 EU foranti-CPS IgG and 100 EU for anti-CPS IgM. The vibriocidal assay wasperformed with V. cholerae O139 isolate SPH1168 as the target strain and2-fold serially diluted sera starting from 1:50 dilution. Thevibriocidal titer is defined as the reciprocal of the highest serumdilution that caused a ≧60% reduction in the number of bacteria comparedto the complement control. Sera from mice injected with saline or CPShad vibriocidal titer <50.

Vibriocidal activity in convalescent sera of cholera patients infectedwith V. cholerae O139 (Table 5). Vibriocidal titers of 20 patient seraranged from 100 to 6400. After absorption with CPS, titers of all seradeclined to ≦50 (baseline for the assay).

Treatment with 2-ME reduced the vibriocidal activity to ≦50 in 17/20sera. The vibriocidal titer of SK 639-2 remained at the same level (400)as found in the untreated serum.

TABLE 5 Serum vibriocidal titers of convalescent sera from patientsinfected with V. cholerae O139 measured before and after absorption withCPS or treatment with 2-mercaptoethanol (2-ME) vibriocidal titer PatientID untreated CPS-absorbed 2-ME-treated SK 391-2 3200 <50 <50 SK 395-21600 <50 <50 SK 428-2 800 <50 <50 SK 456-2 400 <50 <50 SK 458-2 3200 <50<50 SK 494-2 100 <50 <50 5K 504-2 400 <50 <50 SK 522-2 1600 <50 50 SK577-2 1600 <50 <50 SK 591-2 1600 <50 <50 SK 597-2 800 <50 <50 SK 599-23200 <50 <50 SK 622-2 400 <50 50 5K 639-2 400 <50 400 SK 646-2 3200 <50<50 5K 720-2 1600 <50 <50 SK 741-2 800 <50 <50 SK 749-2 >6400 <50 100 SK755-2 1600 <50 200 SK 760-2 1600 <50 50 Each vibriocidal assay wasperformed with 2-fold serially diluted tested serum starting from a1:50-dilution and using V. cholerae O139 SPH1168 as the target strain.

Discussion

Probably because of its complex structure [19, 35] and relatively tightfolded conformation [11], development of synthetic schemes forpreparation of V. cholerae O139 CPS conjugate vaccine was difficult andrequired the use of a readily available protein carrier (chicken serumalbumin, CSA) to optimize the synthetic methods. Slight modifications ofthe two most successful synthetic schemes were then used to prepareconjugates with the medically useful rDT.

Both synthetic schemes involved adipic acid dihydrazide as the linkerand two different activating agents, CDAP and EDC. CDAP was used toprepare AH derivative of CPS (CPS_(AH)), and EDC was used to prepareCSA_(AH) and rDT_(AH). Conjugation of CPS_(AH) with rDT and CSA wasmediated by EDC; alternatively conjugates were prepared by bindingrDT_(AH) and CSA_(AH) with CDAP-activated CPS.

Conjugates such as EDC:CPS-CSA_(AH) and CDAP:CPS-CSA_(AH), althoughprepared from the same components but using different activating agents,are structurally different molecules. EDC activates carboxyls, whileCDAP activates hydroxyls for the reaction with nucleophilic groups [63,64]. In addition, the chemistry of both conjugations is complex becausethe potentially activated groups (carboxyls or hydroxyls) are present onboth CPS as well as CSA_(AH), and they can react with both hydrazidesand amines (ε amine group of lysine) on the protein. It should bepointed out that hydrazides are stronger nucleophiles than amines,therefore, activated carboxyls or hydroxyls will react preferentiallywith hydrazides.

Synthesis of EDC:CPS-CSA_(AH) is representative of several conjugationexperiments: all were accompanied with precipitation of protein, and theresultant conjugates were large in molecular size, had low w/w PS/PRratios (≦0.15), and were poor immunogens. Together these findingsindicate that during EDC-mediated conjugation of CPS and CSA_(AH) theprotein became self-cross-linked, and such structural alteration ofcarrier protein could explain the low immunogenicity of this conjugate.Self-cross-linking of protein could be a direct result of thecomparatively higher reactivity of the CSA-carboxyls than theCPS-carboxyls.

In contrast, no protein precipitation was observed during EDC-mediatedbinding of CPS_(AH) with CSA. In this synthesis the higher reactivity ofprotein carboxyls relative to CPS carboxyls favors the reaction ofprotein carboxyls with they hydrazides of CPS_(AH), which results in theformation of conjugate. The lower reactivity of CPS carboxyls reducesthe extent of self-crosslinking of CPS molecules. Both resultantconjugates, EDC:CPS_(AH1)-CSA and EDC:CPS_(AH2)-CSA, had high PS/PRratios and were significantly better immunogens than EDC:CPS-CSA_(AH).

In contrast to the EDC-mediated coupling of CPS to CSA_(AH), theCDAP-mediated coupling of the same components resulted in the formationof the highly immunogenic conjugate CDAP:CPS-CSA_(AH). It is also ofinterest, that although CSA_(AH) was prepared by an EDC-mediatedderivatization, this exposure to relatively mild conditions (10 mM EDCfor 1 h) had no apparent negative effect on the carrier protein or onthe immunogenicity of the resultant conjugate.

The resultant conjugates elicited serum anti-CPS IgG after the secondinjection and a booster after the third injection when administered tomice by a clinically relevant method and route. Similarly to theimmunologic properties of the V. cholerae O1 serotype Inaba O-specificpolysaccharide conjugates with cholera toxin [10], the V. cholerae O139CPS-CSA and CPS-rDT conjugates elicited high titers of serum vibriocidalantibodies in mice. Treatment with 2-ME reduced (˜4-fold) but did noteliminate the CPS-rDT induced vibriocidal activity, indicating that muchof this activity was mediated by anti-CPS IgG.

I:CPS-rDT_(AH) elicited the highest level of anti-CPS IgG after thethird injection (11.4 EU) but this was not statistically different fromthe levels elicited by II:CPS_(AH)-rDT (10.3 EU) or II:CPS-rDT_(AH)(4.21 EU). On the basis of these data, we plan to clinically evaluateI:CPS-rDT_(AH) and II:CPS_(AH)-rDT.

All four conjugates elicited significant rises of anti-DT IgG after thesecond and third injections. II:CPS_(AH)-rDT elicited the highestpost-third injection level of anti-DT IgG that was significantlydifferent from those of other 3 conjugates (P<0.0007).

I:CPS_(AH)-rDT, prepared by synthesis of CPS_(AH) with rDT at the higherconcentration of EDC (0.05 M), elicited the lowest level of anti-CPSIgG. The level of anti-DT IgG induced by this conjugate was comparableto those elicited by both of CPS-rDT_(AH) indicating that there was nocorrelation between the antibody elicited to the CPS and to the proteincarrier.

There is some confusion about the vibriocidal activity of convalescentsera from patients infected with V. cholerae O139 [3, 17, 25, 28, 40].We found that patient sera convalescent from cholera O139 were uniformlyvibriocidal. The data variation among laboratories may be explained bythe different complement dilutions used for the vibriocidal assays. Wefound that highly diluted complement, used in the vibriocidal assay forV. cholerae O1, is not sufficient to mediate killing of V. cholerae O139which has a capsule. We showed that the undiluted baby rabbit serum, asthe source of complement, is a reliable reagent to demonstrateantibody-initiated lysis of V. cholerae O139.

Similar to the serologic response of humans to the V. cholerae O1infection [1, 24, 29, 32, 34], our results showed that vibriocidalactivity of sera from patients infected with serotype O139 was mostlyspecific to its surface polysaccharide (CPS) and mediated by IgM. Thisis also true for parenterally administered killed whole cell cholera O1vaccine or orally administered attenuated cholera O1 strains [7, 27,44]. In contrast, parenterally administered polysaccharide-proteinconjugate vaccines elicit, in addition to IgM, high levels of serumanti-polysaccharide IgG (2-ME resistant) [10, 38]. We proposed that itis IgG that penetrates on to the intestinal epithelium and initiatescomplement-mediated lysis of the bacterial inoculum and that measurementof the conjugate-induced serum IgG specific to the surfacepolysaccharides of both V. cholerae O1 and O139 should provide areliable method for standardization of these vaccine candidates [36,39].

Diafiltration through YM100 allowed a rapid separation of the lowmolecular weight impurities from V. cholerae O139 CPS. When the materialeluted at Kd 0.91 from SEPHAROSE™ (cross-linked agarose) CL-4B,representing only 1% (by weight) of the unfractionated CPS, wasconcentrated 100-fold and analyzed by SDS-PAGE/Western blot with murinehyperimmune cholera O139 antiserum, it showed two fast-moving bands,similar to that reported for LPS of V. cholerae O139 [4, 45]. Ourresults indicate that diafiltration could be adapted for rapidseparation of CPS and/or LPS from other medically usefulpolysaccharides.

In summary, V. cholerae O139 CPS conjugates with rDT elicited highlevels of serum anti-CPS IgG in mice with vibriocidal activity. Thevibriocidal activity of convalescent sera from patients infected with V.cholerae O139 was mediated mostly by anti-CPS IgM. To verify whether acritical level of anti-CPS IgG will confer immunity to V. cholerae O139,clinical trials of the two most immunogenic CPS-rDT conjugates areplanned.

REFERENCES

1. Benenson, A. S., A. Saad, and W. H. Mosley. 1968. Serological studiesin cholera. 2. The vibriocidal antibody response of cholera patientsdetermined by a microtechnique. Bull. W.H.O. 38:277-285.

2. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 72:248-254.

3. Coster, T. S., K. P. Killeen, M. K. Waldor, D. T. Beattie, D. R.Spriggs, J. R. Kenna, A. Trofa, J. C. Sadoff, J. J. Mekalanos, and D. N.Taylor. 1995. Safety, immunogenicity, and efficacy of live attentuatedVibrio cholerae O139 vaccine prototype. Lancet 345:949-952.

4. Cox, A. D., J-R. Brisson, V. Varma, M. B. Perry. 1996. Structuralanalysis of the lipopolysaccharide from Vibrio cholerae O139. Carbohydr.Res. 290:43-58.

5. Fass, R., M. van de Walle, A. Shiloach, A. Joslyn, J. Kaufman, and J.Shiloach. 1991. Use of high density cultures of Escherichia coli forhigh level production of recombinant Pseudomonas aeruginosa exotoxin A.Appl. Microbiol. Biotechnol. 36:65-69.

6. Finkelstein, R. A. 1962. Vibriocidal antibody inhibition (VAI)analysis: A technique for the identification of the predominantvibriocidal antibodies in serum and for the detection and identificationof Vibrio cholerae antigens. J. Immunol. 89:264-271.

7. Finkelstein, R. A. 1984. Cholera. In: Bacterial Vaccines, Ed. R.Germanier. Academic Press Inc. New York. pp. 107-129

8. Finkelstein, R. A., M. Boesman-Finkelstein, D. K. Sengupta, W. J.Page, C. M. Stanley, and T. E. Phillips. 1997. Colonial opacityvariations among the choleragenic vibrios. Microbiol. 13:23-24

9. Gotschlich, E. C., M. Rey, W. R. Sanborn, R. Triau and B.Cvjetanovic. 1972. The immunological responses observed in field studiesin Africa with Group A meningococcal vaccines. Prog. inImmunobiological. Stand. 129:485-491.

10. Gupta, R. K., D. N. Taylor, D. A. Bryla, J. B. Robbins, and S. C.Szu. 1998. Phase 1 evaluation of Vibrio cholerae O1, serotype Inaba,polysaccharide-cholera toxin conjugates in adult volunteers. Infect.Immun. 66:3095-3099.

11. Gunawardena, A., C. R. Fiore, J. A. Johnson, and C. A. Bush. 1999.Conformation of a rigid tetrasaccharide epitope in the capsularpolysaccharide of Vibrio cholerae O139. Biochemistry. 38:12062-12071.

12. Hall, R. H., F. M. Khambaty, M. H. Kothary, S. P. Keasler, and B. D.Tall. 1994. Vibrio cholerae non-O1 serogroup associated with choleragravis genetically and physiologically resembles O1 El Tor cholerastrains. Infect. Immun. 62:3859-3863.

13. Hoge, Ch. W., L. Bodhidatta, P. Echeverria, M. Deesuwan, and P.Kitporka. 1996. Epidemiologic study of Vibrio cholerae O1 and O139 inThailand: at the advancing edge of the eight pandemic. Am. J. Epidemiol.143:263-268.

14. Inman, J. K., H. M. Dintzis. 1969. The derivatization ofcross-linked polyacrylamide beads. Controlled induction of functionalgroups for the purpose of special biochemical absorbents. Biochem.4:4074-4080.

15. Johnson, V. G., and P. J. Nicholls. 1994. Histidine 21 does not playa major role in diphtheria toxin catalysis. J. Biol. Chem.269:4349-4354.

16. Johnson, J. A., A. Joseph, and J. G. Morris, Jr. 1995. Capsularpolysaccharide-protein conjugate vaccines against Vibrio cholerae O139Bengal. Bull Inst. Pasteur. 93:285-290.

17. Johnson, G., J. Osek, A. M. Svennerholm, and J. Holmgren. 1996.Immune mechanisms and protective antigens of Vibrio cholerae serogroupO139 as a basis for vaccine development. Infect. Immun. 64:3778-3786.

18. Keleti, G., and W. Lederer. 1974. Handbook of micromethods for thebiological Sciences: 3,6-dideoxyhexoses, pp. 57-58. Van NostrandReinhold Company. New York, Cincinnati, Atlanta, Dallas, San Francisco,London, Toronto, Melbourne.

19. Knirel, Y. A., L. Paredes, P-E. Jansson, A. Weintraub, G. Widmal andM. J. Albert. 1995. Structure of the capsular polysaccharide of Vibriocholerae O139 synonym Bengal containing D-galactose-4,5-cyclophosphate.Eur. J. Biochem. 232:391-396.

20. Kohn, J., M. Wilchek. 1983. 1-Cyano-4-dimethylaminopyridiniumtetrafluoroborate as a cyanylating agent for the covalent attachment ofligand to polysaccharide resins. FEBS Letts. 154:209-210.

21. Konadu, E., J. Shiloach, D. A. Bryla, J. B. Robbins, S. C. Szu.1996. Synthesis, characterization and immunological properties in miceof conjugates composed of detoxified lipopolysaccharide of Salmonellaparatyphi A bound to tetanus toxoid, with emphasis on the role ofO-acetyls. Infect. Immun. 64:2709-2715.

22. Kossaczka, Z., S. Bystricky, D. A. Bryla, J. Shiloach, J. B.Robbins, and S. C. Szu. 1997. Synthesis and immunological properties ofVi and Di-O-acetyl pectin protein conjugates with adipic aciddihydrazide as the linker. Infect. Immun. 65:2088-2093.

23. Lees, A., B. L. Nelson, and J. J. Mond. 1996. Activation of solublepolysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroboratefor use in protein-polysaccharide conjugate vaccines and immunologicalreagents. Vaccine 14:190-198.

24. Levine, M. M., D. R. Nalin, J. P. Craig, D. Hoover, E. J. Bergquist,D. Waterman, H. P. Holley, R. B. Hornick, N. P. Pierce and J. P.Libonati. 1979. Immunity of cholera in man: Relative role ofantibacterial versus antitoxic immunity. Trans. Royal Soc. Trop. Med.Hyg. 73:3-9.

25. Losonsky, G. A., Y. Lim, P. Motamedi, L. Comstock, J. A. Johnson, J.G. Morris, Jr., C. O. Tacket, J. B. Kaper, and M. M. Levine. 1997.Vibriocidal antibody responses in North American volunteers exposed towild-type or vaccine Vibrio cholerae O139: Specificity and relevance toimmunity. Clin. Diagnos. Lab. Immunol. 4:264-269.

26. Meno, Y., M. K. Waldor, J. J. Mekalanos, and K. Amako. 1998.Morphological and physical characterization of the capsular layer ofVibrio cholerae O139. Arch. Microbiol. 170:339-344.

27. Merritt, C. B., and R. B. Sack. 1970. Sensitivity of agglutinatingand vibriocidal antibodies to 2-mercaptoethanol in human cholera. J.Infect. Dis. 121:S25-S30.

28. Morris, J. G., G. E. Losonsky, J. A. Johnson, C. O. Tacket, J. P.Nataro, P. Panigrahi, and M. M. Levin. 1995. Clinical and immunologiccharacteristics of Vibrio cholerae O139 Bengal infection in NorthAmerican volunteers. J. Infect. Dis. 171:903-908.

29. Mosley, W. H. 1969. The role of immunity in cholera. A review ofepidemiological and serological studies. Tex. Rep. Biol. Med. 27(Suppl1):227-241.

30. Nandy, R. K., M. J. Albert, A. C. Ghose. 1996. Serum antibacterialand antitoxin responses in clinical cholera caused by Vibrio choleraeO139 Bengal and evaluation of their importance in protection. Vaccine14:1137-1142.

31. Nandy, R. K., S. Mukhopadhyay, A. N. Ghosh, and A. C. Ghose. 1999.Antibodies to the truncated (short) form of “O” polysaccharides (TFOP)of Vibrio cholerae O139 lipopolysaccharides protect mice againstexperimental cholera and such protection is mediated by inhibition ofintestinal colonization of vibrios. Vaccine. 17:2844-2852.

32. Neoh, S. H., and D. Rowley. 1980. The antigens of Vibrio choleraeinvolved in the vibriocidal action of antibody and complement. J.Infect. Dis. 121:505-513.

33. Pike, R. M., and C. H. Chandler. 1971. Serological properties of Gand M antibodies to the somatic antigen of Vibrio cholerae during thecourse of immunization of rabbits. Infect. Immun. 6:803-809.

34. Plikaytis, B. D., P. F. Holder, and G. M. Carlone. 1996. ProgramELISA for Windows. User's Manual 12, Version 1.00. Centers for DiseaseControl, Atlanta, Ga.

35. Preston, L. M., Q. Xu, J. A. Johnson, A. Joseph, D. R. Maneval Jr,K. Hussain, G. P. Reddy, C. A. Bush, and J. G. Morris Jr. 1995.Preliminary structure determination of the capsular polysaccharide ofVibrio cholerae O139 Bengal A11837. J. Bacteriol. 177:835-838.

36. Robbins, J. B., R. Schneerson and S. C. Szu. 1995. Perspective:Hypothesis: Serum IgG antibody is sufficient to confer protectionagainst infectious diseases by inactivating the inoculum. J. Infect.Dis. 171:1387-1398.

37. Schneerson, R., O. Barrera, A. Sutton, and J. B. Robbins. 1980.Preparation, characterization and immunogenicity of Haemophilusinfluenzae type b polysaccharide-protein conjugates. J. Exp. Med.152:361-376.

38. Sengupta, D. K., M. Boesman-Finkelstein, and R. A. Finkelstein.1996. Antibody against the capsule of Vibrio cholerae O139 protectsagainst experimental challenge. Infect. Immun. 64:343-345.

39. Szu, S. C., R. Gupta, and J. B. Robbins. 1994. Induction of serumvibriocidal antibodies by O-specific polysaccharide-protein conjugatevaccines for prevention of cholera. p. 381-394. In I. K. Wachsmuth, P.A. Blake and O Olsvik (ed). Vibrio cholerae. American Society forMicrobiology, Washington D.C.

40. Tacket, C. O., G. E. Losonsky, J. P. Nataro, L. Comstock, J.Michalski, R. Edelman, J. B. Kaper, M. M. Levine. 1995. Initial clinicalstudies of CVD 112 Vibrio cholerae O139 live oral vaccine: safety andefficacy against experimental challenge. J. Infect. Dis., 172:883-886.

41. U.S. Department of Health and Human Services, Public Health Service,Food and Drug Administration Guideline on Validation of the LimulusAmebocyte Lysate Test As an End-product Endotoxin Test for Human andAnimal Parenteral Drugs, Biological Products, and Medical Devices. 1987.

42. Waldor, K. M., J. J. Mekalanos. 1994. Emergence of a new cholerapandemic: molecular analysis of virulence determinants in Vibriocholerae O139 and development of a live vaccine prototype. J. Infect.Dis., 170:278-283.

43. Waldor, M. K., R. Colwell, and J. J. Mekalanos. 1994. The Vibriocholerae O139 serogroup antigen includes an O-antigen capsular andlipopolysaccharide virulence determinants. Proc. Natl. Acad. Sci (USA)91:11388-11392

44. Wasserman, S. G., G. A. Losonsky, F. Noriega, C. O. Tacket, E.Castaneda and M. M. Levine. 1994. Kinetics of the vibriocidal antibodyresponse to live oral cholera vaccines. Vaccine. 11:1000-1003.

45. Weintraub, A., G. Widmalm, P. -E. Jansson, M. Jansson, K. Hultenby,and M. J. Albert. 1994. Vibrio cholerae O139 Bengal possesses a capsularpolysaccharide which may confer increased virulence. Microb. Pathog.16:235-241.

46. Oscarson, S., U. Tedebark, and D. Tuerk. 1997. Synthesis ofcolitose-containing oligosaccharide structures found in polysaccharidesfrom Vibrio cholerae O139 synonym Bengal using thioglycoside donors.Carbohydr. Res. 299:159-164.

47. Kossacza, Z., J. Shiloach, V. Johnson, D. N. Taylor, R. A.Finkelstein, J. B. Robbins, and S. C. Szu. 2000. Vibrio cholerae O139Conjugate Vaccines: Synthesis and Immunogenicity in Mice of V. choleraeO139 Capsular Polysaccharide Conjugates with Recombinant DiphtheriaToxin Mutant in Mice. Infect. Immun. 68:5037-5043.

48. For reviews, see:

-   -   (a) J. B. Robbins, R. Schneerson, S. Szu, V. Pozsgay, In:        Vaccinia, vaccinations and vaccinology: Jenner, Pasteur and        their successors (Ed.: S. Plotkin, B. Fantini), Elsevier, Paris,        1996, p. 135-143.    -   (b) R. K. Sood, A. Fattom, V. Pavliak, R. B. Naso, Drug        Discovery Today 1996, 1:381-387.    -   (c) A. Fattom, Adv. Expt. Med. Biol. 1995, 383:131-139.    -   (d) U. B. S. Sφrenson, Danish Med. Bull. 1995, 42:47-53.    -   (e) H. J. Jennings, R. K. Sood, In Neoglycoconjugates.        Preparation and Applications (Eds. Y. C. Lee, R. T. Lee),        Academic Press, New York, 1994, pp. 325-371.    -   (f) W. Egan, Ann. Rep. Med. Chem. 1993, 28:257-265.    -   (g) P. R. Paradiso, K. Dermody, S. Pillai, Vaccine Research        1993, 2:239-248.    -   (h) H. J. Jennings, Curr. Top. Microbiol. Immunol. 1990,        150:97-127.

49. For the development of this concept, see:

-   -   (a) K. Landsteiner, The specificity of serological reactions,        Harvard University Press, Cambridge, 1970.    -   (b) W. F. Goebel, O. T. Avery, J. Exp. Med. 1929, 50:521-531.

50. J. B. Robbins, R. Schneerson, P. Anderson, D. H. Smith, J. Am Med.Assoc. 1996, 276:1181-1185.

51. For example:

-   -   (a) D. Cohen, S. Ashkenazi, M. S. Green, M. Gdalevich, G.        Robin, R. Slepon, M. Yavzori, N. Orr, C. Block, Y. Ashkenazi, J.        Schemer, D. N. Taylor, T. L. Hale, J. D. Sadoff, D.        Pavliakova, R. Schneerson, J. B. Robbins, Lancet, 1997,        349:155-0159.    -   (b) D. Cohen, S. Ashkenazi, M. S. Green, Y. Lerman, R.        Slepon, G. Robin, N. Orr, D. N. Taylor, J. C. Sadoff, C. Chu, J.        Shiloach, R. Schneerson, J. B. Robbins, Infect. Immun. 1997,        64:4074-4077.

52. Fournier, J. M., S. Villeneuve. 1998. Actualite du cholera etproblematique vaccinale [Cholera update and vaccination problems]. Med.Trop. 58 (2 Suppl): 32-35.

53. V. P. Bondre, V. B. Sinha, B. S. Srivastava. 1998. Evaluation ofdifferent subcellular fractions of Vibrio cholerae O139 in protection tochallenge in experimental cholera. FEMS Imm. Med. Micro. 19:323-329.

54. Fattom, A., C. Lue, S. C. Szu, J. Mestecky, G. Schiffman, D. A.Bryla, W. F. Vann, D. Watson, L. M. Kimzey, J. B. Robbins, and R.Schneerson. 1990. Serum antibody response in adult volunteers elicitedby injection of Streptococcus pneumoniae type 12F polysaccharide aloneor conjugated to diphtheria toxoid. Infect. Immun., 58:2309-2312.

55. Devi, S. J., J. B. Robbins and R. Schneerson. 1991. Antibodies topoly[(2→8)-α-N-acetylneuraminic acid] are elicited by immunization ofmice with Escherichia coli K92 conjugates: Potential vaccines for groupsB and C meningococci and E. coli. Proc. Natl. Acad. Sci. USA88:7175-7179.

56. Szu, S. C., X. Li, R. Schneerson, J. H. Vickers, D. Bryla, and J. B.Robbins. 1989. Comparative immunogenicities of Vi polysaccharide-proteinconjugates composed of cholera toxin or its B subunit as a carrier boundto high- or lower-molecular-weight Vi. Infect. Immun. 57:3823-3827.

57. Szu, S. C., X. Li, A. L. Stone, and J. B. Robbins. 1991. Relationbetween structure and immunologic properties of the Vi capsularpolysaccharide. Infect. Immun. 59:4555-4561.

58. Szu, S. C., A. L. Stone, J. D. Robbins, R. Schneerson, and J. B.Robbins. 1987. Vi capsular polysaccharide-protein conjugates forprevention of typhoid fever. J. Exp. Med. 166:1510-1524.

59. Szu, S. C., D. N. Taylor, A. C. Trofa, J. D. Clements, J. Shiloach,J. C. Sadoff, D. A. Bryla and J. B. Robbins. 1994. Laboratory andpreliminary clinical characterization of Vi capsularpolysaccharide-protein conjugate vaccines. Infect. Immun. 62:4440-4444

60. C. Chu. B. Liu, D. Watson, S. Szu, D. Bryla, J. Shiloach, R.Schneerson and J. B. Robbins. 1991. Preparation, Characterization, andImmunogenicity of Conjugates Composed of the O-Specific Polysaccharideof Shigella dysenteriae Type 1 (Shiga's Bacillus) Bound to TetanusToxoid. Infect. Immun., 59:4450-4458.

61. Robbins, J. B.; R. Schneerson, S. C. Szu, D. A. Bryla, F. Y. Lin, E.C. Gotschlich. 1998. Standardization may suffice for licensure ofconjugate vaccines. Dev. Biol. Stand. 95:161-167.

62. Favre, D., S. J. Cryz Jr., J. -F. Viret. 1996. Construction andCharacterization of a Potential Live Oral Carrier-Based Vaccine againstVibrio Cholerae O139. Infect. Immun. 64:3565-3570.

63. Shafer D. E., B. Toll, R. F. Schuman, B. L. Nelson, J. J. Mond, A.Lees 2000. Activation of soluble polysaccharides with1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use inprotein-polysaccharide conjugate vaccines and immunological reagents.II. Selective crosslinking of proteins to CDAP-activatedpolysaccharides. Vaccine 18:1273-1281

64. Hermanson, G. T. 1996. Bioconjugate techniques, Academic Press, SanDiego.

Modifications of the above described modes for carrying out theinvention that are obvious to those of skill in the fields ofimmunology, protein chemistry, medicine, and related fields are intendedto be within the scope of the following claims.

Every reference cited hereinabove is hereby incorporated by reference inits entirety.

1. A conjugate molecule, comprising the capsular polysaccharide ofVibrio cholerae O139, covalently bound with an adipic acid dihydrazidelinker to a carrier protein, wherein the carrier protein is arecombinant diphtheria toxin comprising CRMH21G, wherein the conjugatecomprises a polysaccharide to protein ratio of about 0.76 and elicitsserum antibodies vibriocidal to Vibrio cholerae O139.
 2. Apharmaceutical composition comprising the conjugate molecule of claim 1,in a physiologically acceptable carrier.
 3. A method of eliciting serumantibodies in a mammal that have vibriocidal activity against Vibriocholerae O139, comprising conjugating a capsular polysaccharide ofVibrio cholerae O139 covalently bound with an adipic acid dihydrazidelinker to a carrier protein, in which the carrier protein is arecombinant diphtheria toxin comprising CRMH21G, by about 0.02 M1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), thereby preparing aconjugate molecule comprising a polysaccharide to protein ratio(weight/weight) of about 0.76; administering to the mammal at leastthree doses of a therapeutically effective amount of the conjugatemolecule, thereby eliciting serum antibodies comprising anti-diphtheriatoxin IgG at a concentration of about 1050 ELISA units/milliliter ofserum.
 4. A method of immunizing a mammal against Vibrio cholerae O139,comprising administering to the mammal a therapeutically effectiveamount of an isolated antibody or fragment thereof, wherein the antibodyis elicited by the method of claim
 3. 5. The method of claim 3, whereinthe mammal is a human.
 6. The method of claim 3, wherein the conjugatemolecule is administered at a dose of about 1 microgram to about 100milligrams of Vibrio cholerae O139 capsular polysaccharide.
 7. Themethod of claim 4, wherein the mammal is a human.
 8. The method of claim4, wherein the isolated antibody is administered at a dose of about 1mg/kg of body weight to about 10 mg/kg of body weight.